WO2012129115A2 - Substances et procédés utilisables en vue du profilage de micro-arn - Google Patents

Substances et procédés utilisables en vue du profilage de micro-arn Download PDF

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WO2012129115A2
WO2012129115A2 PCT/US2012/029522 US2012029522W WO2012129115A2 WO 2012129115 A2 WO2012129115 A2 WO 2012129115A2 US 2012029522 W US2012029522 W US 2012029522W WO 2012129115 A2 WO2012129115 A2 WO 2012129115A2
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universal
sequence
mirna
primer
molecules
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PCT/US2012/029522
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WO2012129115A3 (fr
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Shyam S. Mohapatra
Jia-Wang Wang
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University Of South Florida
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Priority to US14/000,517 priority Critical patent/US9493825B2/en
Publication of WO2012129115A2 publication Critical patent/WO2012129115A2/fr
Publication of WO2012129115A3 publication Critical patent/WO2012129115A3/fr
Priority to US14/962,984 priority patent/US20160160274A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • Micro RNAs typically 18 to 25nt in length, are non-protein-coding RNAs that can inhibit the translation of target mRNAs (Croce and Calin. 2005. miRNAs, cancer, and stem cell division. Cell 122(1): 6-7). miRNAs directly or indirectly regulate a wide range of genes, and are involved in a remarkable spectrum of biological pathways including cell development, proliferation and apoptosis (He and Harmon. 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7): 522-31 , Alvarez-Garcia and Miska. 2005. MicroRNA functions in animal development and human disease. Development 132(21): 4653-62).
  • miRNA entries from vertebrates, flies, worms, plants, and viruses including 721 human miRNAs and 579 mouse miRNAs, have been annotated (miRBase, Release 14) in the Sanger Institute miRNA sequence database (Griffiths-Jones, Saini, van Dongen and Enright. 2008. miRBase: tools for microRNA genomics. Nucleic Acids Res 36(Database issue): D 154-8); the function of many miRNAs is unknown.
  • miRNAs are difficult to detect because they are relatively short nucleic acid molecules (on average, only about 22 bases in length).
  • these short sequences can be present in sequences other than mature miRNA, such as pre-miRNA, pri-miRNA, genomic DNA and mRNA.
  • pre-miRNA pre-miRNA
  • pri-miRNA genomic DNA
  • mRNA it is difficult to distinguish mi NAs within the same family, as these miRNAs usually differ from each other only in terms of one or a few nucleotides.
  • Tm melting temperatures
  • RNA 11(9): 1461 -70 Nelson, Baldwin, Scearce, Oberholtzer, Tobias and Mourelatos. 2004. Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1(2): 155-61). These techniques are not as sensitive or specific, when compared to quantitative real-time reverse transcription PGR ( qRT-PCR).
  • RT-qPCR reverse transcriptase quantitative polymerase chain reaction assays
  • SYBR Green detects all forms of nucleic acids, including double-stranded DNA, double- stranded RNA, single-stranded RNA and DNA, although tire detection sensitivity of double- stranded RNA, single-stranded RNA and DNA is lower than that of double-stranded DNA.
  • the cost of making hundreds of target-specific probes and RT primers during miRNA screening tests can be prohibitive.
  • the Chen et al. method is procedurally complex. Profiling each miRNA requires an RT reaction; otherwise, if only one RT reaction is performed, all miRNA-specific RT primers need to be mixed together. Further, hundreds of target-miRNA specific TaqMan® probes need to be added separately in order to detect or quantify miRNA.
  • RT primers used in the Chen et al. method only have a 6-nt-sequence that base-pairs with the target miRNAs.
  • the RT primers may hybridize to, and prime, other RNAs during the RT reaction (Tang, Hajkova, Barton, Lao and Surani. 2006. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 34(2):e9). Accordingly, improved methods for profiling miRNAs are needed. BRIEF SUMM ARY OF THE INVENTION
  • the present invention provides materials and methods for detecting, quantifying, and/or profiling microRNAs.
  • the present invention is sensitive, specific, convenient, and cost-effective.
  • the present invention can detect mature miRNA in a sample that comprises non-mature miRNA nucleic acids (such as one or more of genomic DNA, pre-miRNA, pri-miRNA and mRNA, and cDNAs thereof) that also comprise the target miRNA sequence.
  • materials for detecting, quantifying, and/or profiling microRNAs comprise: a universal primer for reverse transcription of miRNAs, a universal reverse primer for PCR amplification reaction, and a universal probe (multiple universal probes can be used to increase the detection sensitivity). Also provided are reagents and kits for detecting, quantifying, and/or profiling miRNAs.
  • the universal primer for reverse transcription is an oligonucleotide comprising: a (dT) n sequence flanked by a stem-looped universal adaptor sequence, wherein "n" is an integer ranging from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3 ' end of the (dT) n sequence, and the nucleotide immediately adjacent to the (dT) constitute sequence is not T, and wherein the universal adaptor sequence near the 5 ' end of the (dT) n sequence forms a stem- loop structure by base- pairing.
  • the universal reverse primer is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of the adaptor sequence near the 5 * end of the (dT)n sequence.
  • the universal probe comprises a sequence that is, or base-pairs with, at least part, of the adaptor sequence near the 5' end of the (dT) n sequence.
  • the universal primer for reverse transcription comprises SEQ
  • the universal reverse primer for PGR amplification comprises SEQ ID NO: 1.
  • the universal probe comprises SEQ ID NO: 3.
  • the present invention provides assays for detecting, quantifying, and/or profiling miRNAs.
  • the present invention can detect a plurality of target miRNAs using one reverse transcription reaction and one qPCR reaction.
  • the present invention can detect, quantify, and/or profile miRNAs at a level of about 1 pg of total RNA.
  • the method for detecting, quantifying, and/or profiling a target miRNA comprises :
  • amplification reaction e.g., PGR
  • the present invention uses Surveyor nuclease and/or single strand endonucleases that remove mis-matches caused by mispriming to enhance specificity and/or single strand endonucleases such as Exonuclease I that remove single- stranded DNA such as excess primers.
  • the present invention uses uracil-DNA Glycosylase (UDG) and/or dlJTP to enhance specificity.
  • a plurality of probes is used for detection and/or quantification of target miRNAs.
  • the universal probe comprises one or more locked- nucleic acids (LNAs).
  • the forward primer comprises the target-miRNA sequence, one or more additional nucleotides (such as adenine molecules) attached to the 3 ' end of the target mature miRNA sequence, and one or more additional nucleotides attached to the 5' end of the target mature miRNA sequence.
  • additional nucleotides such as adenine molecules
  • FIG. 1 is a schematic illustration of an embodiment of the qRT-PCR assay for profiling miRNA, using a universal TaqMan® probe and a universal RT primer.
  • miRNAs are polyadenylated using poly(A)polymerase molecules.
  • the poly(A)-tailed miRNAs are then reverse-transcribed into cDNAs using a universal primer comprising oligo dTs flanked by a stem-loop adaptor sequence.
  • the cDNAs are amplified by qRT- PCRs using a mature target miR A sequence as the forward primer and a universal reverse primer (QRTU).
  • QRTU universal reverse primer
  • a universal TaqMan® probe is used to detect the amplification product.
  • Q-Quencher such as lABlk_FQ (Iowa BlackTM FQ)
  • F Fluorescent Dye, such as FAM (6-carboxyfluorescein).
  • SEQ ID NOS: 27-30 are sequences shown
  • Figure 2 shows characteristics of the universal probe and RT primer (UPR) miRNA real-time quantitative PCR (qRT-PCR) assay.
  • A Amplification curve plot of mi R -690 UQmiR qRT-PCR assay.
  • B Relative standard curve plot of miR-690 UQmiR qRT-PCR assay calculated over 10 serial dilutions. Average CT values (y-axis) are plotted against the logarithm of the input amount of RNA (x-axis) added to each sample. Standard curve of the slope was -3.4 and correlation coefficient was 0.9995.
  • C Correlation of total RNA input to the threshold of cycle (CT) values in four miRNA assays.
  • CT threshold of cycle
  • FIG. 3 shows that the UPR Q-RT-PCR assay is miRNA-specific.
  • A Amplification curve plot of the UPR miRNA qRT-PCR assay, using total RNA. as the template for reverse transcription without the step of polyadenylation (mRNA).
  • mRNAs are used as templates for amplification of let-7, miR-21, miR-142, miR-150 and miR-494, the cycle threshold values (Cts) of qRT-PCRs are greater than 38.
  • NTCs nontemplate controls
  • the 11-4 and 11-18 amplifications of the cDNA molecules serve as positive controls of the RT reaction.
  • Mouse tail genomic DNA (DNA) and transcribed spleen total RNA (miRNA, served as positive controls) were subjected to polyadenylation and reverse transcription reactions. There are no detectable signals for the DNA template.
  • C Amplification curve plot of UPR miRNA qRT-PCR assay using mouse tail genomic DNA as the template of the 96 m iRNA qPCR array.
  • FIG. 4 shows characteristics of the SYBR Green real-time quantitative PCR (qRT- PCR) assay for 96 miRNAs.
  • the SYBR Green Q-PCRs were performed using SYBR Green.
  • a forward primer specific to the target miRNA and a universal reverse primer were used for qPCR amplification.
  • FIG 5 is a schematic illustration of an embodiment of the UQmiR quantitative realtime PCR assay for profiling miRNAs, using one or more universal hydrolysis probes and a universal RT primer.
  • miRNAs are polyadenylated using poly( A polymerase molecules.
  • the poly(A)-tailed miRNAs are then reverse-transcribed into cDNAs using a universal primer comprising oligo-dTs flanked by a stem-loop adaptor sequence (see Table 2).
  • the cDNAs are amplified by RT-qPCR using a UQmiR forward primer comprising the target mature miRNA sequence and a universal reverse primer (QRTU).
  • the forward primer (UQmiR primer) of the amplification reaction comprises the target mature miRNA sequence and additional sequences attached to the 5'- and 3 '-ends of the mature miRNA sequence.
  • the UQmiR-primer is annealed to the cDNA of the target mature miRNA, and extension occurs at both ends of the primer-cDNA complex; however, unlike the complex formed by the UQmiR-primer and the mature miRNA cDNA, there is no extension at both ends of the complex formed by the UQmiR-primer and other DNA molecules.
  • Attachment of additional sequences at the 5 '- and 3 ' ends of the target mature miRNA sequence also results in higher Tin for the UQmiR primer.
  • the Tm of the cDNA template from a specific miRNA may be increased.
  • the UQmiR primer does not anneal to, or has a low probability of annealing to, the nucleic acid molecules other than cDNA from mature miRNA (such as, genomic DNA, cDNAs of pre-miRNA, pri- miRNA and mRNA) that contain the mature miRNA sequence; Due to lower homology between the UQmiR primer and those molecules, the complex formed by them is less stable than the complex formed by the UQmiR primer and the cD A from specific mature miRNA.
  • mature miRNA such as, genomic DNA, cDNAs of pre-miRNA, pri- miRNA and mRNA
  • Figure 7 compares the sensitivity of the RT-qPCR reaction between the UQmiR assay of the present invention and the commercially-available miRNA RT-qPCR Taqman assay (Applied Biosystems (ABl)). Same amounts of synthetic miR-142 and Let-7a mature miRNAs were used for both miRNA RT-qPCR assays following the manufacturer's instruction. The results show that the UQmiR assay of the present invention is at least two times more sensitive than the commercially-available Taqman assay.
  • Figures 8A-B show that the UQmiR miRNA qPCR assay of the present invention discriminates mature miRNAs from pre-miRNAs.
  • Mature miRNA-specific and pre-miRNA- specific PCR primers which have extra sequences at the 3 " -ends but no extra sequences at the 5 " -ends, were used to detect pre-miRNA.
  • a total of 7.85E8 copies of synthetic pre-rniR- 142 and pre-miR- 150 were added to the RT reaction.
  • the results show that mature miRNA- specific primers do not amply pre-miRNAs.
  • the folds of discrimination are over 50 times.
  • the Y axis represents fluorescence intensity corresponding to the specific amplification levels of the template.
  • Figures 9A-C show that the UQmiR miRNA qPCR assay of the present invention discriminates mature miRNAs from pre-miRNAs.
  • UQmiR primers consisting of sequences from mature miRNA and extra sequences that flank the mature miRNA sequences were used to detect mature miRNA and pre-miRNA sequences.
  • a total of 7.85E8 copies of synthetic Let-7a, miR-142, pre-I .ei-7a. pre -miR-142 and pre-miR-150 were added to the RT reaction.
  • the results show that mature miRNA-specific primers do not amply pre-miRNAs. The folds of discrimination are over one thousand times.
  • Figure 10 shows that the UQmiR miRNA qPCR assay of the present invention discriminates mouse Let- 7 miRNA family members.
  • Four closely-related let-7 family members (let-7a to let-7d) were used in the assay.
  • Relative miRNA detection percentages (%) to the perfectly matched targets were calculated based on Cq values.
  • a total of 7.85E8 copies of synthetic RNA were added to the RT reaction.
  • the numbers in parentheses indicate the difference in bases between each pair of miRNAs.
  • Figure 11 shows relative plasma miRNA expression levels of human plasma miRNAs pooled from five normal volunteers measured by the RT-qPCR miRNA assay of the present invention.
  • the results show that the present RT-qPCR miRNA assay can sensitively and specifically detect 88 out of 94 miRNAs (those miRNAs have high expression levels in mouse spleens from the dot blot array results) in a sample that only contains 0.8 ml of plasma for each miRNA.
  • Figure 12 shows that the UQmiR miRNA qPCR assay of the present invention discriminates mature miRNAs from pre-miRNAs.
  • Mature miRNA-specific primers were used to detect mature miRNA and pre-miRNA sequences.
  • a total of 7.85E8 copies of synthetic pre-miR-142 and pre-miR-150 were added to the RT reaction.
  • the results show that mature miRNA-specific primers do not amply pre-miRNA. The folds of discrimination are over one thousand times.
  • Figure 13 shows discrimination of mature miRNA and pre-miRNA using the UQmiR miRNA qPCR assay with Surveyor nuclease, Exonuclease I nuclease and UQmiR primer that only has 3 '-end extra sequence.
  • UQmiR primers containing mature miRNA sequences were used to detect mature miRNA and pre-miRNA sequences.
  • a total of 7.85E8 copies of synthetic Let-7a, miR-142, pre-I.et-7a. pre-miR-142 and pre-miR-150 were added to RT reaction.
  • the results show that UQmiR primer for detecting mature miRNA primers do not detect pre-miRNA well.
  • the folds of discrimination are over one thousand times, which are shown above the each bar.
  • Figure 14 shows discrimination UDG assay of mouse Let-7 miRNA family members.
  • Four closely related let-7 family members (from let-7a to let-7 d) were included in the assay.
  • Relative miRNA detection percentages (%) to the perfectly matched targets were calculated based on Cq values.
  • a total of 7.85E8 copies of synthetic RNA were added to the RT reaction.
  • the numbers in parentheses indicate different bases between each pair of the miRNAs.
  • RT primer that contains multiple Uracil nucleotides were used (LUTVN RT primer and Ublocker in Table 2), and after the double strand cDNA synthesis, a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VII I was used to remove LUTVN RT primer and Ublocker. Very low levels of non-specific signal were observed, ranging from 0 to 0.18% (0 to 0.3, Chen et al. method) for miRNAs with 2-5 mismatched bases and only 0.1 1 to 0.88% (0.1 to 3.7, Chen et al. method) for the miRNAs that differed by a single nucleotide.
  • Figure 15 illustrates an embodiment of UQmiR primers designed for the Let -7 miRNA family.
  • UQmiR primers are designed so that they have minimal homology between each other, especially within a miRNA family, to increase UQmiR specificity between homologous miR A sequences. The number of different bases between each miRNA sequence is greatly increased from one or a few.
  • mmu-let ⁇ 7a SEQ ID NO: 19
  • mmu-let-7c SEQ ID NO:20
  • mmu-let-7b SEQ ID NO:21
  • mmu-let-7d SEQ ID NO:22
  • mmu-let-7e SEQ ID NO:23
  • mmu-let-7f SEQ ID NO:24
  • mmu-let-7g SEQ ID NO:25
  • mmu-let-7i SEQ ID NO: 26
  • SEQ ID NO: 1 is a universal primer sequence for reverse transcription of miRNAs oop).
  • SEQ II) NO: 2 is a universal reverse primer sequence in qPCR reaction (QRTU ).
  • SEQ ID NO: 3 is a universal probe sequence (miRU probe).
  • SEQ ID NO: 4 is a forward primer sequence for qPCR reaction (Let-7a).
  • SEQ II) NO: 5 is a forward primer sequence for qPCR reaction (miR-21 ).
  • SEQ II) NO: 6 is a forward primer sequence for qPCR reaction (miR-142).
  • SEQ ID NO: 7 is a forward primer sequence for qPCR reaction (miR-150).
  • SEQ I D NO: 8 is a forward primer sequence for qPCR reaction (miR-494).
  • SEQ ID NO: 9 is a forward primer sequence for qPCR reaction (mmu-miR-690).
  • SEQ ID NO: 10 is a universal primer sequence for reverse transcription of miRNAs.
  • SEQ ID NO: 11 is a universal reverse primer sequence in qPCR reaction.
  • SEQ ID NO: 12 is a Ublocker sequence useful according to the present invention.
  • SEQ ID NO: 13 is a universal primer sequence for reverse transcription of miRNAs.
  • SEQ ID NO: 14 is a universal reverse primer sequence in qPCR reaction.
  • SEQ ID NO: 15 is a universal reverse primer sequence in qPCR reaction.
  • SEQ ID NO: 16 is a universal hydrolysis LNA probe sequence.
  • SEQ ID NO: 17 is a universal hydrolysis LNA probe sequence.
  • SEQ ID NO: 18 is a universal hydrolysis LNA probe sequence.
  • SEQ ID NO: 19 is a primer sequence designed for the mmu-let ⁇ 7a miRNA useful according to the present invention (Fig. 15).
  • SEQ ID NO: 20 is a primer sequence designed for the mmu-let-7c miRNA useful according to the present invention (Fig. 15).
  • SEQ ID NO: 21 is a primer sequence designed for the mmu-let-7b miRNA useful according to the present invention (Fig. 15).
  • SEQ 11) NO: 22 is a primer sequence designed for the mmu-let-7d miRNA useful according to the present invention (Fig. 15).
  • SEQ ID NO: 23 is a primer sequence designed for the mmu-let-7e miRNA useful according to the present invention (Fig. 15).
  • SEQ ID NO: 24 is a primer sequence designed for the mmu-let-7f miRNA useful according to the present invention (Fig. 15 .
  • SEQ ID NO: 25 is a primer sequence designed for the mmu-let-7g miRNA useful according to the present invention (Fig. 15).
  • SEQ ID NO: 26 is a primer sequence designed for the mmu-let-7i miRNA useful according to the present invention (Fig. 15).
  • SEQ ID NO: 27 is a poly(A) sequence (Fig. 1).
  • SEQ ID NO: 28 is a poly(T) cDNA sequence reverse-transcribed from a poly(A) sequence (Fig. 1).
  • SEQ ID NO: 29 is a poly(A) sequence (Fig. 1 ).
  • SEQ II) NO: 30 is a poly(A) sequence (Fig. 1).
  • the present invention provides materials and methods for detecting, quantifying, and/or profiling microRNAs.
  • the present invention uses a reverse transcription reaction and a PCR amplification reaction.
  • the present invention uses a universal probe, such as a TaqMan® probe, and a universal RT-primer (UPR).
  • a universal probe such as a TaqMan® probe, and a universal RT-primer (UPR).
  • the UQmiR qRT-PCR assay can be sensitive, specific, convenient, and cost-effective.
  • the present invention uses a universal probe (such as a TaqMan ⁇ ) and a universal RT primer (UPR).
  • the present invention can detect a plurality of target miRNAs using one RT reaction and a single universal probe (multiple universal probes may be used to increase the detection sensitivity).
  • the present invention allows detection and quantification of miRNAs in as little as Ipg total RNA.
  • the present invention can delect mature miRNA in a sample that contain non-mature miRNA nucleic acids (such as genomic DNA, pre-miRNA, pri- miRNA and mRNA, and cDNAs thereof) that also comprise the target miRNA sequence.
  • non-mature miRNA nucleic acids such as genomic DNA, pre-miRNA, pri- miRNA and mRNA, and cDNAs thereof
  • genomic DNA and mRNA in total RNA samples produce no or little detectable signals.
  • Total RNA used in the present invention can be obtained from simple extraction methods, such as, Trizol extraction.
  • Total RNA samples used in the present invention need not be treated with DNases or undergo small RNA fractionation or purification, which are not only labor intensive procedures, but also may result in significant loss of input miRNAs.
  • the miRNA UQmiR qRT-PCR assay of the present invention has two major advantages when compared to the conventional miRNA stem-loop qRT-PCR methods (such as the Chen et al. method).
  • a highly specific universal poly (T) primer for example with a stretch of 25Ts, is used to prime the RT reaction for detection of all target miRNAs.
  • the conventional methods use a stem-loop RT primer that has a 6nt sequence specific to each target miRNA sequence.
  • the 25Ts poly (T) sequence in the universal primer theoretically appears only once in a random sequence of 1.1259E+15bps, while the 6nt -sequence in the miRNA-specific primer appears 652962 times in a random sequence of the mouse genomic size.
  • the 6nt, target-miRNA-specific primer used in the prior art methods is not genome-wide specific; It can not only prime the target miRNAs, but also prime other RNAs that have the 6nt sequences.
  • the present invention can use a universal probe (such as a universal TaqMan® probe) for the detection and qualification of a plurality of miRNAs.
  • a universal probe such as a universal TaqMan® probe
  • the conventional methods require target-miRNA-specific TaqMan® probes, where each probe can only detect one target miRNA.
  • conventional methods require that each target-specific probe need to be individually added each time.
  • the present invention also embodies the use of multiple universal probes to increase the detection sensitivity.
  • the present invention provides a RT-qPCR miRNA Taqman assay (UQmiR) for profiling miRNAs.
  • UQmiR RT-qPCR miRNA Taqman assay
  • the present invention also involves use of computer programs (such as QmiR) for detection, quantification and/or profiling of miRNAs.
  • the RT-qPCR miR A assay comprises one RT reaction with one universal RT primer, one universal reverse primer for the amplification reaction, miRNA-specific UQmiR forward primers, and one or more universal hydrolysis probes to sensitively detect all miRNAs (Figure 5 and Table 2).
  • the UQmiR forward primers only detect mature miRNA sequences, and do not amplify non mature miRNA sequences, such as genomic DNA, cD As of pre-miRNA, pri-miRNA and niRNA ( Figure 6).
  • the present invention also comprises the use of a computer program, such as the QmiR computer program, to enhance specificity of the RT-qPCR miRNA Taqman assay (UQmiR) assay in the detection, quantification and/or profiling of miRNAs.
  • a computer program such as the QmiR computer program
  • the present invention uses Surveyor nuclease that remove mis-matches caused by misprinting to enhance specificity and/or single strand endonucleases such as Exonuclease I that remove single strand DNA such as excess primers.
  • the present invention uses uracil-DNA Glycosylase (UDG) and/or dUTP to enhance specificity.
  • UDG can be used to destroy the templates of any non-specific amplification when use dL I P-containing oligos as RT primers.
  • Figures 13-15 describe various embodiments of using Surveyor nuclease, endonucleases, uracil-DNA Glycosylase (UDG) and/or dUTP for enhancing specificity and sensitivity.
  • One aspect of the present invention provides materials for detecting, quantifying, and/or profiling miRNAs.
  • the materials comprise: a universal primer for reverse transcription of miRNAs, a universal reverse primer for PCR amplification reaction, and a universal probe.
  • reagents and kits for detecting, quantifying, and/or profiling miRNAs are also provided.
  • the present invention provides a universal primer for reverse transcription of miRNAs.
  • the universal primer is an oligonucleotide sequence comprising: a (dT) n sequence flanked by a stem-looped universal adaptor sequence, wherein n is an integer ranges from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3 'end of the (dT) nest sequence, and the nucleotide immediately adjacent to the (dT) n sequence is not T, and wherein the universal adaptor sequence near the 5 'end of the (dT) n sequence forms into a stem-loop structure by base -pairing.
  • the universal primer for reverse transcription is single-stranded DNA.
  • n is 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, n can be an integer less than 10 or greater than 50.
  • the universal primer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or
  • the adaptor sequence comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 56, 57, 58, 59, or 60 nucleotides. In some embodiments, the adaptor sequence comprises more than 60 nucleotides. In some embodiments, each stem of the adaptor sequence located near the 5 " end of the (dT) makeup sequence comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides. In one embodiment, the stems of the adaptor sequences base pair with each other (see Figures 1 and 5).
  • each stem of the adaptor sequence located near the 5 'end of the (dT) radical sequence comprises more than 25 nucleotides.
  • the loop of the adaptor sequence comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides, hi certain embodiments, the loop of the adaptor sequence can comprise more than 25 nucleotides. In one embodiment, the loop of the adaptor comprises single-stranded nucleotides.
  • Figures 1 and 5 illustrate embodiments of the universal primer for reverse transcription of miRNAs.
  • the universal primer for reverse transcription of miRNAs comprises SEQ ID NO: 1.
  • the universal primer for reverse transcription of miRNAs comprises SEQ ID NO: 10 or SEQ ID NO: 13 .
  • the adaptor sequence (near the '5 end) of the universal primer for reverse transcription of miRNAs does not comprise a sequence that hybridizes, or base- pairs, with the target miRNA, wherein the sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 20 nucleotides in length.
  • the universal primer for reverse-transcription is not target miRNA specific. Design of Universal Reverse Primers for PCR Amplification Reaction
  • the present invention provides a universal reverse primer for PCR amplification reaction.
  • the universal reverse primer is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of the adaptor sequence (located near the 5 " end of the (dT) n sequence) of the universal primer for reverse transcription.
  • the universal reverse primer is not target miRNA specific.
  • the universal reverse primer is single-stranded DNA.
  • the universal reverse primer for PCR amplification comprises 8 ,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22. 23, 24, or 25 nucleotides. In some embodiments, the universal reverse primer for PCR amplification comprises less than 8 or more than 25 nucleotides.
  • Figures 1 and 5 illustrate embodiments of the universal reverse primer for PCR amplification reaction.
  • the universal reverse primer for PCR amplification reaction comprises SEQ ID NO; 2.
  • the universal reverse primer for PCR amplification does not comprise a sequence that is, or base-pairs with, the target miRNA sequence. In one embodiment, the universal reverse primer for PCR amplification is not target miRNA specific.
  • the universal reverse primer for PCR amplification comprises one or more dUTPs. In some embodiments, the universal reverse primer for PCR amplification reaction comprises SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the present invention provides a mature miRNA-specific forward primer for PCR amplification reaction.
  • the forward primer is miRNA- specific.
  • the forward primer comprises at least part of the target miRNA sequence.
  • the forward primer comprises the entire target miRNA sequence.
  • the forward primer is single-stranded DNA.
  • the forward primer comprises at least 6, 7, 8, 9, 10, 1 1 , 12, 13,
  • the forward primer comprises one or more additional nucleotides (such as adenine molecules) attached to tire 3' end of the target mature miRNA sequence.
  • additional nucleotides such as adenine molecules
  • 1 , 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides are attached to the 3 ' end of the target mature miRNA sequence.
  • the forward primer can detect the target miRNA sequence other than non mature miRNA sequences (e.g., genomic DNA and cDNAs of pre-miRNA, pri -miRNA) with higher specificity.
  • cDNAs of pre-miRNA, pri-miRNA and mRNA sequences and genomic DNA sequences do not extend at their 3 '-end although they may anneal to the UQmiR forward primer comprising the additional nucleotides (such as adenine molecules).
  • the forward primer comprises one or more additional nucleotides attached to the 5 ' end of the target mature miRNA sequence.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 1 7, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30additional nucleotides are attached to the 5 ' end of the target mature miRNA sequence.
  • Tm is about 60 °C to 75 °C, 60 °C to 72 °C, 65 °C to 72 °C, 67 °C to 72 °C, or 69 °C to 71 °C. In some embodiments, Tm is about 65 °C, 66 °C, 67 °C, 68°C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74°C, and/or 75 °C. Preferably, Tm is about primer 70 °C.
  • variation of Tm of the forward primer is with in 0.5 °C, 1 °C, 1.5 °C, 2 °C, 2.5 °C, 3 °C, 3.5 °C, 4 °C, 4.5 °C, 5 °C, 5.5 °C, 6 °C, 6.5 °C, 7 °C, 8 °C, 9 °C, or 10 °C.
  • Figures 1 , 5 and 6 illustrate embodiments of the forward primer for PGR amplification reaction.
  • the present invention provides a universal probe.
  • the universal probes are useful for the detection and/or quantification of target miRNAs.
  • the universal probe is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of the adaptor sequence of the universal primer for reverse transcription.
  • the universal probe is single-stranded DNA.
  • the universal probe further comprises a fluorophore, or other detectable moiety, attached at the ends of the oligonucleotide.
  • the universal probe in PGR amplification comprises 3, 4, 5,
  • the universal probe can comprise more than 25 nucleotides.
  • a plurality of probes can be used for detection and/or quantification of target miRNAs.
  • the universal probe comprises one or more locked-nucleic acids (LNAs).
  • the universal probe comprises a sequence that is, or base- pairs with, at least part of a stem -looped adaptor sequence near the 5 " end of the (dT)n sequence of the adaptor sequence, wherein the probe comprises one or more locked nucleic acids.
  • Figures 1 and 5 illustrate embodiments of the universal probe.
  • the universal probe comprises SEQ ID NO: 3.
  • the universal probe comprise SEQ ID NO: 16, 17 or 18.
  • the universal probe does not comprise a sequence that is, or base-pairs with, the target miRNA sequence. In one embodiment, the universal probe is not target miRNA specific.
  • the detectable moiety is preferably a fluorophore.
  • the fluorophores are preferably attached to the ends or near the ends of the oligonucleotide.
  • the probe undergoes a conformational change to bring the fluorophores closer in proximity to each other. This change in distance causes a change in the photon absorption or emission of the fluorophores, creating a visual indication that the probe of the invention has bound a target sequence.
  • Fluorescent resonance energy transfer (FRET) or non-FRET interactions are used to detect the binding of the probe to its target sequence (e.g., PGR amplification products).
  • FRET interactions also known as non -radiative energy transfer; see Yaron e! al.. Analytical Biochemistry 95:228-235 (1979)
  • FRET interactions for quenching fluorescence signals requires spectral overlap between the donor and acceptor fluorophore moieties and the efficiency of quenching is directly proportional to the distance between the donor and acceptor moieties of the FRET pair. Extensive reviews of the FRET phenomenon are described in Clegg, R. M., Methods Enzymol., 221 : 353-388 (1992) and Selvin, P.
  • the probe When the probe binds to the target sequence, the probe will undergo a conformational change causing the distance and/or angle between the fluorophore pairs to change. This change can then be detected because it will change the efficiency of resonance energy transfer between the fluorophore moieties after exposure of the probe to an excitation wavelength of light.
  • fluorophores useful according to the present invention include, but are not limited to, FAM (6-carboxyfluorescein), CY5, CY3, BODIPY FL, and TEXAS RED.
  • FAM (6-carboxyfluorescein)
  • CY5 CY3, BODIPY FL
  • TEXAS RED a fluorophore
  • the universal primer, probe and adaptor sequences can be derived from universal probe and primer sequences known in the art, such as universal TaqMan® probe and primer sequences.
  • the design of the stem-loop universal adaptor sequence is described in (Chen, Ridzon, Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar, Andersen, Lao, Livak and Guegler. 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20): el 79).
  • oligonucleotides of the present invention can encompass single and double- stranded RNA, single and double-stranded DNA and cD.NA. nucleic acid analogs, aptamers, and the like.
  • nucleic acid and “oligonucleotide” are used interchangeably herein.
  • the oligonucleotide strands of the probe are single-stranded DNA.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between a particular purine and a particular pyrimidine in double-stranded nucleic acid molecules (DNA-DNA, DNA-RNA, or RNA-RNA).
  • the major specific pairings are guanine with cytosine and adenine with thymine or uracil.
  • Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like.
  • hybridization is conducted under high stringency conditions by techniques well known in the art, as described, for example, in Keller, G.H. & M.M. Manak, DNA Probes, and the companion volume DNA Probes: Background, Applications, Procedures (various editions, including 2 nd Edition, Nature Publishing Group, 1993). Hybridization is also described extensively in the Molecular Cloning manuals published by Cold Spring Harbor Laboratory Press, including Sambrook & Russell, Molecular Cloning: A Laboratory Manual (2001). Each of these publications is incorporated herein by reference in its entirety.
  • a non-limiting example of high stringency conditions for hybridization is at least about 6X SSC and 1 % SDS at 65 "C, with a first wash for 10 minutes at about 42 °C with about 20% (v/v) form amide in 0.1X SSC, and with a subsequent wash with 0.2X SSC and 0.1 % SDS at 65 °C.
  • a non-limiting example of hybridization conditions are conditions selected to be about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 °C lower than the thermal melting point (T m ) for the specific sequence in the particular solution.
  • T m is the temperature (dependent upon ionic strength and pi I ) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • T m typically increases with [Na " ] concentration because the sodium cations electrostatically shield the anionic phosphate groups of the nucleotides and minimize their repulsion.
  • the washes employed may be for about 5, 10, 15, 20, 25, 30, or more minutes each, and may be of increasing stringency if desired.
  • the melting temperature may be described by the following formula (Beltz, G.A., K.A. Jacobs, T.H. Eickbush, P.T. Cherbas, and F.C. Kafatos, Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285, 1983).
  • Tm 81.5°C + 16.6 Log[Na+] + 0.41 (%G+C) - 0.61(%formamide) - 600/length of duplex in base pairs.
  • T m may be obtained using nearest-neighbor models. Breslauer, et al, Proc. Natl. Acad. Sci. USA, 83:3746-3750 (1986); SantaLucia, Proc. Natl Acad. Sci. USA, 95: 1460-1465 (1998); Allawi & SantaLucia, Biochemistry 36: 10581 -94 (1997); Sugimoto et al, Nucleic Acids Res., 24:4501-4505 (1996).
  • T m may also be routinely measured by differentia] scanning calorimetry (Duguid et al., Biophys J, 71 :3350-60, 1996) in a chosen solution, or by other methods known in the art, such as UV-monitored melting. As the stringency of the hydridization conditions is increased, higher degrees of homology are obtained.
  • kits for detecting, quantifying, and/or profiling miRNA comprises a universal primer for reverse transcription, a universal reverse primer for an amplification reaction (e.g. , PCR), and/or a universal probe.
  • the kit can further comprise a forward primer sequence for an amplification reaction (e.g., PCR).
  • the kit comprises SEQ ID NO: l and/or SEQ ID NO:2. In another specific embodiment, the kit further comprises SEQ ID NO:3. In a further specific embodiment, the kit comprises one or more of SEQ ID NOs: 4-9. In a specific embodiment, the kit comprises one or more of SEQ ID NO:s 1 -18.
  • the kit may include any material useful for performin any step of the present invention.
  • the kit may further comprise any material useful for reverse transcription of RNAs, and/or for profiling the target K A.
  • the kit may poly(A)polymerases, dNTPs, Adenosine-5'-triphosphates (ATP), DNA ligases (e.g., T4 DNA ligase), and Taq DNA polymerase.
  • the kit may also comprise, e.g. , a buffering agent, a preservative, or a stabilizing agent.
  • a buffering agent e.g., a preservative, or a stabilizing agent.
  • Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions (e.g., printed instructions).
  • Another aspect of the present invention provides an assay for detecting, quantifying, and/or profiling miRNAs.
  • the present invention can detect, quantify, and/or profile miRNAs in a sample of about 1 pg.
  • the method for detecting, quantifying, and/or profiling a target miRNA comprises:
  • an amplification reaction e.g., PGR
  • the universal primer for reverse transcription is an oligonucleotide comprising: a (dT) n sequence flanked by a stem-looped universal adaptor sequence, wherein n is an integer ranges from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3 'end of the (dT) n sequence, and the nucleotide immediately adjacent to the (dT) counsel sequence is not T, and wherein the universal adaptor sequence near the 5 'end of the (dT sequence forms into a stem-loop structure by base-pairing, wherein the universal reverse primer for the amplification reaction is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of the adaptor sequence of the universal primer for reverse transcription.
  • the forward primer comprises the target-miRNA sequence, one or more additional nucleotides (such as adenine molecules) attached to the 3 ' end of the target mature miRNA sequence, hi some embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides (such as adenine molecules) are attached to the 3 ' end of the target mature miRNA sequence, hi one embodiment, the forward primer further comprises one or more additional nucleotides attached to the 5 ' end of the target mature miRNA sequence. In some embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, or 9 additional nucleotides are attached to the 5 ' end of the target mature miRNA sequence.
  • the assay comprises the steps of:
  • a plurality of probes can be used for detecting and/or quantifying a level of amplified c-DNA molecules.
  • 2, 3, 4, 5, 6, or 7 probes are used for detecting and/or quantifying a level of amplified c-DNA molecules.
  • the universal probe comprises a detectable moiety, for example, a fluorophore.
  • a plurality of detectable moieties e.g., fluorophores can be used.
  • the amplification reaction e.g., PCR
  • qRT-PCR quantitative real time polymerase chain reaction
  • the PCR reaction is performed once. In another embodiment, the reverse transcription reaction is performed once.
  • the miRNA profiling assay of the present invention uses one universal primer for reverse transcription, one universal reverse primer in the amplification reaction (e.g., PCR), and/or one universal probe. In one embodiment, the miRNA profiling assay detects, quantifies, and/or profiles a plurality of target miRNAs.
  • the threshold cycle (Ct) of the PCR amplification reaction ranges from 15 to 37.
  • the Ct value of the PCR amplification reaction ranges from 17 to 35, 20 to 30. 23 to 33, or 25 to 30.
  • the Ct value is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, or 37.
  • the Ct value does not exceed 38.
  • the UQmiR qRT-PCR assay can be used for detecting, quantifying, and/or profiling nucleotide molecules, such as for example, siRNAs, oligonucleotides, polynucleotides, and/or mRNAs.
  • samples containing miRNAs is a total RNA sample.
  • the sample contains DNA, or has not been treated with DNases, or both.
  • the samples can be derived from an organism, including mammals such as apes, chimpanzees, orangutans, humans, monkeys; and domesticated and/or laboratory animals such as dogs, cats, horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs, and hamsters.
  • the samples can be derived from, including but not limited to, a sample containing tissues, cells, and/or biological fluids isolated from a subject.
  • Embodiment 1 A method for detecting, quantifying, and/or profiling a target miRNA, comprising:
  • the universal primer for reverse transcription is an oligonucleotide comprising: a (dT) n sequence ilanked by a stem-looped universal adaptor sequence, wherein "n” is an integer ranging from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3 'end of the (dT) nest sequence, and the nucleotide immediately adjacent to the (dT) n sequence is not T, wherein the universal adaptor sequence near the 5 'end of the (dT) n sequence forms into a stem-loop structure by base-pairing, and
  • the universal reverse primer in the amplification reaction is an oligonucleotide comprising a sequence that is, or base-pahs with, at least part of the adaptor sequence near or toward the 5 'end of the (dT) n sequence.
  • Embodiment 2 The method, according to embodiment 1 , wherein the forward primer comprises the target miRNA sequence and three or more additional nucleotides attached to the 3 ' end of the target mature miRNA sequence.
  • Embodiment 3 The method, according to embodiment 1, wherein the additional nucleotides attached to the 3' end of the target mature miR A sequence are adenine molecules.
  • Embodiment 4 The method, according to embodiment 2, wherein one or more additional nucleotides are attached to the 5 ' end of the target mature miRNA sequence.
  • Embodiment 5 The method, according to any of the preceding embodiments, wherein the amplification reaction for cDNA amplification is quantitative real time polymerase chain reaction (qRT-PCR).
  • qRT-PCR quantitative real time polymerase chain reaction
  • Embodiment 6 The method, according to any one of embodiments 1 -5, comprising:
  • the universal probe comprises a sequence that is, or base-pairs with, at least part of the adaptor sequence near the 5 'end of the (dT) n sequence.
  • Embodiment 7 The method, according to embodiment 6, wherein the universal probe comprises locked nucleic acid (LNA) molecules.
  • LNA locked nucleic acid
  • Embodiment 8 The method, according to embodiment 6 or 7, wherein the universal probe includes a detectable moiety.
  • Embodiment 9 The method, according to embodiment 8, wherein the detectable moiety is a tluorophore selected from FAM, CY5, CY3, BODIPY FL, TEXAS RED, or any combination of two or more of the foregoing.
  • the detectable moiety is a tluorophore selected from FAM, CY5, CY3, BODIPY FL, TEXAS RED, or any combination of two or more of the foregoing.
  • Embodiment 10 The method, according to any one of embodiments 6-9, wherein at least two universal probes are used.
  • Embodiment 11 The method, according to any one of the preceding embodiments, wherein the amplification reaction for amplification of cDNA molecules is performed once.
  • Embodiment 12 The method, according to any one of the preceding embodiments, wherein the threshold cycle (Ct) of the amplification reaction for amplification cDNA molecules ranges from 17 to 35.
  • Embodiment 13 The method, according to any one of the preceding embodiments, wherein the sample is a total RNA sample.
  • Embodiment 14 The method, according to any one of the preceding embodiments, wherein the method is capable of detecting, quantifying and/or profiling microRNA in a total RNA sample of about 1 pg.
  • Embodiment 15 The method, according to any one of the preceding embodiments, wherein the universal reverse primer comprises dUTP.
  • Embodiment 1 6 The method, according to embodiment 15, further comprising adding uracil-DNA Glycosylase molecules to the amplification reaction mixture.
  • Embodiment 17 The method, according to any one of the preceding embodiments, comprising adding nuclease molecules to the reaction mixture.
  • Embodiment 18 A universal primer for detecting, quantifying and/or profiling miRNA, wherein the universal primer is a primer for reverse transcription of miRNAs, wherein the universal primer is an oligonucleotide comprising: a (dT) n sequence flanked by a stem-looped universal adaptor sequence, wherein "n" is an integer ranging from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3 'end of the (dT) n sequence, and the nucleotide immediately adjacent to the (dT) n sequence is not T, and wherein the universal adaptor sequence near the 5 'end of the (dT) n sequence fonns into a stem -loop structure by base-pairing.
  • Embodiment 19 A universal primer for detecting, quantifying and/or profiling miRNA, wherein the universal primer is a reverse primer for an amplification reaction, wherein the universal primer is an oligonucleotide comprising a sequence that is, or base- pairs with, at least part of the adaptor sequence near the 5'end of the (dT) n sequence according to embodiment 18.
  • Embodiment 20 The universal reverse primer for an amplification reaction according to embodiment 19, wherein the universal reverse primer comprises dU I .
  • Embodiment 21 A universal probe for detecting, quantifying and/or profiling miRNA, wherein the universal probe comprises a sequence that is, or base-pairs with, at least part of the adaptor sequence near the 5'end of the (dT) n sequence of embodiment 18.
  • Embodiment 22 The universal probe according to embodiment 21 , further comprising a detectable moiety.
  • Embodiment 23 The universal probe according to embodiment 21 or 22, wherein the detectable moiety comprises a fluorophore.
  • Embodiment 24 The universal probe according to any one of embodiments 21- 23, comprising locked nucleic acid (LNA) molecules.
  • LNA locked nucleic acid
  • Embodiment 25 A kit for detecting, quantifying and/or profiling a level of expression of miRNA, comprising a universal primer for reverse transcription according to embodiment 18; and a universal reverse primer for an amplification reaction according to embodiment 19 or 20.
  • Embodiment 26 The kit according to embodiment 25, further comprising a universal probe of any one of embodiments 21-24.
  • Table 1 illustrates oligonucleotide sequences useful according to the present invention.
  • miR-494 TGAAACATACACGGGAAACCTCTT (SEQ ID NO: 8)
  • mmu-miR-690 AAAGGCTAGGCTCACAACCAAA (SEQ ID NO: 9)
  • N * A, G, or C
  • N ** A, G, C, or T.
  • ABBREVIATION S miRNAs: MicroRNAs; UPR: a universal probe and RT primer; qRT- PGR: quantitative real-time reverse transcription PGR; QRTU: universe reverse primer: RTUloop: universal RT primer; FAM: 6-carboxyiluorescein; CT: threshold cycle.
  • NewURP GCCTCTGACTCCAGGATCTGTAGAC (SEQ ID NO:l 1 )
  • LNADLP 1 [6FAM] ACC[ A] [T] [C] A[ A] [T] [C] G[T] G[T]G [BHQ 1 ] * * *
  • N A, G, or C
  • N ' * A, G, C, or T. ** The bases in parenthesis are special bases called LNAs (locked nucleic acids).
  • FIG. 1 illustrates one embodiment of the universal probe-RT primer (UPR) realtime qRT-PCR assay for profiling microRNAs.
  • the assay comprises a three-step process. First, total RNA is polyadenylated using poly(A)polymerase molecules. The polyadenylated miRNAs are reversely transcribed into cDNAs using a universal RT primer (RTUloop) with 25-T poly (T) at 3 ' end. Finally, the RT product is amplified by qPCR using the mature target miRNA sequence as the forward primer and a universal reverse primer (QRTU). The amplification products can be detected or quantified using a miRU fluorescent probe, such as for example, a 26-nucleotide universal TaqMan® probe.
  • a miRU fluorescent probe such as for example, a 26-nucleotide universal TaqMan® probe.
  • miRNA expression profiling was performed and analyzed using the dot-blot array as previously described (Wang and Cheng. 2008. A simple method for profiling miRNA expression. Methods Mol Biol 414: 183-90), 96 mi R As were selected for miRNA QPCR array assay from the dot blot array results.
  • the miRNA universal TaqMan® probe was designed by PrimerQuest (Table 1 ) based on sequences described in Ro et al.
  • the stem-loop adaptor contained in the RT primers were designed according to Chen et al. (Chen, Ridzon, Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar, Andersen, Lao, Livak and Guegler. 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20 ): el 79). All DNA oligonucleotides were synthesized by IDT. Preparation of RNA and cDNA
  • Total RNAs ( ⁇ g) was incubated with poly(A)polymerase (USB) molecules, thereby generating polyadenylated microRNAs at the '3 end.
  • the polyadenylated RNAs (20 ⁇ ) reverse transcribed using 2 ⁇ RTUloop primer, 0.25 mM each of dNTPs, 100 units Smartscribe reverse transcriptase, I x reverse transcriptase buffer, and 10 mM DTT (Clontech Laboratories). The reactions were incubated at 42°C for 90 minutes, and then at 95°C for 5 minutes, to inactivate reverse transcriptase molecules and to degrade RNAs. All reverse transcriptase reactions included no-template and minus-RT as controls.
  • Amplification curves were generated with an initial denaturing step at 95°C for 30 seconds, followed by 40 cycles at 95°C for 5 seconds, and at 60°C for 30 seconds.
  • Dissociation curves were generated using the following programs: PCR products were denatured at 95°C for 15 seconds, cooled to 60°C for 15 seconds, and finally at 95°C for 15 seconds.
  • qRT-PCR cU A templates of miRNAs were prepared by 10-time serial dilution, which is equivalent to 10, 1 , 0.1 , 0.01 and O.OOlng of total RNA, respectively. Each sample was run in triplicate.
  • the standard curves were generated using the Prism HT7900 system.
  • the correlation coefficient (R2) values were greater than 0.99 (except for that of miR-150, which is 0.98), indicating excellent linear reliability between RNA concentration and the CT value of reverse transcription real-time PGR reaction for each miRNA.
  • Figures 2A&B show the amplification curve plot and standard curve plot of the miR-690 qRT-PCR.
  • the PGR efficiency is 0.94 for miR-690 and 1.1 for miR- 142 and miR- 150 (efficiencies between 0.90 and 1.10 are typically acceptable).
  • RNA isolation method can consistently produce RNA free from genomic DNA contamination. This Example tests whether residual genomic DNA contamination may cause non-specificity of the UPR miRNA qRT-PCR assay.
  • mouse tail genomic DNA isolated by the DNeasy Blood & Tissue Kit
  • the results show no detectable signals in the UPR miRNA QPCR assay, when using genomic DNA either as a sham control for the total RNA ( Figure 3B), or as the direct template of QPCR ( Figure 3CC).
  • the agarose gel electrophoresis shows nonspecific amplification ( Figure 3D), which may be detected by SYBR Green.
  • the amount of the genomic DNA used in this Example far exceeds residual DNA that may be present in total RNA preparation. This indicates that the accuracy of the UPR miRNA qRT-PCR assay will not he affected by genomic DNA contamination in total RNA preparation.
  • Total RNA was also used as the template of UPR miRNA qRT-PCR assay without the polyadenylation step. This investigates whether mRNAs, the major component of total R A, will produce significant non-specific signals in the assay.
  • CTs threshold cycle
  • CT values greater than 35 approaches the sensitivity limits of the real-time PCR detection system of miRNAs. This suggests that the contribution of the background signals from mRNA in this assay is negligible.
  • SYBR Green is a fluorescent dye. It non-spec ifically intercalates into double- stranded DNA and detects double-stranded DNA.
  • Figure 4 shows detection of non-specific amplification by 96-miRNA qRT-PCR array- using SYBR Green.
  • the dissociation curves ( Figure 4A) show that most miRNA qRT-PCRs produced a small peak and a major peak. The small peak represents non-specific amplification, while the major peak represents amplification of the desired PCR products.
  • the log amplification curve plot ( Figure 4B) shows that there are non-exponent amplifications for low abundant miRNAs. The results suggest that the SYBR Green assay may over-detect miRNAs, which is present in low amounts.
  • LNA hydrolysis locked-nucleic Acid
  • Figure 5 multiple hydrolysis locked-nucleic Acid (LNA) probes are used to sensitively detect amplicons of the amplification reaction.
  • LNA hydrolysis locked-nucleic Acid
  • Use of a plurality of probes significantly enhances sensitivity for miRNA profiling and the increase in sensitivity equals to the number of probes used minus 1.
  • This technique is especially useful for detecting low copy of miRNAs and/or detecting miRNAs in small samples.
  • miRNAs present in samples obtained from laser capture microdissection microscopy (LCMM) or a single cell can be detected using the present probe design. Such low copy of miRNAs cannot be detected usmg the commercially-available miRNA detection methods.
  • FIG. 7 shows that the UQmiR assay of the present invention is more sensitive than the Taqman RT-qPCR assay, which is the most sensitive and specific miRNA assay currently available.
  • the forward primer (UQmiRprimer) useful for the amplification reaction of the present miRNA assay.
  • the forward primer comprises the target mature miRNA sequence and additional nucleotides attached to t he 5 ' and 3 ' ends of the mature miRNA sequences.
  • the UQmiR primers specifically bind to the cDNAs of mature miRNAs and extension occurs at both ends.
  • the forward primer does not anneal to non mature miRNA sequences such as genomic DNA, cDNAs of pre-miRNA, pri-miRNA and mRNA that comprise the mature miRNA sequence; therefore, no extension occurs in non mature miRNA sequences.
  • UQmiR primers that comprise extra sequences at the 3 ' -end of the miRNA sequence discriminate mature miRNAs over pre-miRNAs in 50 folds (Figure 8A-B).
  • UQmiR primers that comprise extra sequences at both 3 '- and 5 ' -ends of the mature miRNA sequence discriminate mature miRNAs over pre-miRNAs over one thousand times; thus, the present miRNA assay is far more specific than the ABI miRNA Taqman assay (Figure 9A-C).
  • FIG 10 shows that the RT-qPCR miRNA Taqman assay (UQmiR) assay discriminates miRNAs within the mouse Let-7 miRNA family.
  • the miRNA members of the same family are highly homologous and can differ from each other only in terms of a few bases.
  • the UQmiR miRNA qPCR assay is as sensitive as the commerciallyavailable
  • the UQmiR can sensitively detect plasma and serum miRNAs in clinical samples.
  • Figure 12 shows that the present miRNA assay sensitively and specifically detect
  • Protocol a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3: 12.

Abstract

La présente invention concerne des substances et des procédés utilisables en vue de la détection, de la quantification et/ou du profilage haut débit de micro-ARN. La présente invention présente l'avantage de se révéler plus sensible et plus spécifique que d'autres analyses de PCR quantitative pour miARN actuellement disponibles. En outre, la présente invention est commode, facile à mettre en œuvre et économique. Dans un mode de réalisation, la présente invention concerne une amorce universelle pour transcription inverse de tous les miARN, une amorce antisens universelle pour réaction d'amplification par PCR et des sondes universelles. Dans un autre mode de réalisation, la présente invention concerne des tests permettant une détection et/ou une quantification simultanée d'une pluralité de miARN cibles dans le cadre d'une unique réaction de transcription inverse.
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